芦笋废弃资源的生物炼制
文献类型：学位论文
| 作者 | 赵庆生 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05-15 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 赵兵 |
| 关键词 | 芦笋 废弃资源 生物炼制 叶绿素铜钠盐 芦笋多糖 生物农药 |
| 其他题名 | Biorefinery of asparagus (Asparagus officinalis L.) waste |
| 学位专业 | 生物化工 |
| 中文摘要 | 芦笋（Asparagus officinalis L.）是一种药食同源的名贵蔬菜，我国年产鲜笋≥140万吨。芦笋的初加工产品主要有罐头、速冻食品、芦笋干以及芦笋粉等。在芦笋加工过程中约有占原料总量30～40%的笋皮、老茎等下脚料产生，这些下脚料除了极少一部分作为动物饲料，大部分被丢弃任其自然腐败，既污染了环境，又造成了资源的极大浪费。据测定，这些下脚料中含有丰富的营养物质和活性成分，有些成分的含量甚至高于蔬菜笋，而现有的单一组分利用技术存在能耗高、经济性差、固体废弃物处理等问题。为了解决芦笋废弃资源利用过程中急需解决的上述问题，本研究以生物炼制为思路，首先分级提取芦笋叶绿素、芦笋多糖、芦笋皂苷和β-胡萝卜素等，并进一步合成了绿色食用色素－叶绿素铜钠盐，分离纯化了芦笋多糖，研究了其单糖组成、理化性质及生物活性；将提取芦笋皂苷产生的残渣固态发酵、鲜芦笋下脚料液态发酵制备Bt生物农药，为实现芦笋废弃资源的高值化全利用奠定了基础。本论文得到的主要研究结果如下： 1、在单因素实验的基础上，优化了芦笋叶绿素的超声强化提取工艺，在提取溶剂为80%丙酮: 95%乙醇= 1:4，提取时间60 min，提取温度70℃，料液比1:10，超声功率160 W条件之下，芦笋鲜叶中叶绿素提取率为0.287%。得到了叶绿素铜钠盐合成的最佳皂化条件为：pH 11，皂化时间60 min，皂化温度60℃，浓缩5倍。采用100 W超声处理30秒，或200 W超声20秒，可显著促进叶绿素酮酸的聚集，使之易于采用抽滤或离心的方式回收。优化的酸化、铜代的条件为pH 3，时间2 h，硫酸铜实际用量为理论用量的1.5倍，温度70℃，以鲜叶计产品得率为0.18%（鲜叶含水量62.2%），经检测产品质量符合国家标准GB 3262-1982。叶绿素铜钠盐合成过程所产生的废液经Al2O3柱层析可分离回收β-胡萝卜素，β-胡萝卜素总含量（以鲜叶计）为0.0161%，柱层析后回收率为49.3%，纯度为90%。干燥后的芦笋残渣制备芦笋粗多糖得率为13.3%，其中多糖含量为20.1%；沉淀多糖的乙醇溶液通过蒸馏回收，浓缩膏干燥后得粗芦笋皂苷，皂苷得率为12.3%，含量为14.23%。 2、在单因素实验的基础上，采用Box Benhnken设计和响应面分析法，设计3因素3水平实验，得到芦笋粗多糖提取的优化工艺条件为：超声功率700 W，占空比1.5:1，提取时间45 min，料液比1:35。以纯多糖的得率为响应值，获得了芦笋多糖得率对编码自变量超声功率（X1）、提取时间（X2）、料液比（X3）的二次多项回归方程： Y=3.04 + 0.23X1 + 0.19X2 + 0.16X3 + 0.057X1X2 – 0.00425X1X3 – 0.06 X2X3 – 0.74 – 0.14 – 0.21 并按照模型给出的最优提取条件：超声功率 600 W，占空比1.5:1，提取时间 46 min，液固比 35 mL/g，验证实验的多糖得率为3.134%，与预测值接近，验证了模型的正确性。与热水提取法相比，超声循环提取法具有时间短、得率高、低能耗的优点。超声强化提取对多糖的分子量没有影响，而热水提取的芦笋多糖有明显的降解，表现为其电泳迁移率较大。对芦笋多糖进行了脱色、脱蛋白、分级沉淀等处理，测定了芦笋多糖分子量，分析了芦笋多糖的组分及理化特性。芦笋粗多糖经纯化，纯度提高到72%。采用40%、60%、80%乙醇沉淀所得的芦笋多糖，分子量分别为57539、52296和51061。芦笋多糖由葡萄糖、岩藻糖、阿拉伯糖、果糖、鼠李糖（2.18: 1.86: 1.50: 0.98: 1.53）组成。芦笋多糖的红外图谱显示的吸收峰均为多糖特有的吸收峰，芦笋多糖具有β-型糖苷键。芦笋多糖对羟基自由基(·OH)、DPPH自由基以及超氧阴离子自由基( )都有一定的清除效果，其中40%乙醇沉淀得到的芦笋多糖对羟基自由基的消除作用接近Vc。芦笋多糖对Hela和BEL-7404细胞有显著的抑制作用，并且具有明显的剂量依赖性。1%浓度的芦笋粗多糖，72小时对Hela细胞和BEL-7404细胞的抑制率分别为54.64%、57.31%。初步纯化后的芦笋多糖对Hela细胞和BEL-7404细胞的抑制率分别为67.86%，57.73%，其中40%乙醇沉淀的芦笋多糖对Hela细胞的抑制率达83.96%，60%乙醇沉淀的芦笋多糖对BEL-7404细胞的抑制率达64.18%。因此，芦笋多糖可作为保健品及抗癌辅助药物的原料药来开发。 3、芦笋提取皂苷产生的残渣固态发酵Bt生物农药的最佳条件为：发酵温度30℃，湿度85%，培养基初始pH值7.4，通气量1～1.2 vvm，干发酵产物芽孢数为1.55×1011 cfu/gDW，蛋白含量为8.83 mg/gDW。鲜芦笋下脚料在10 L发酵罐液态发酵Bt生物农药的最优条件为初始pH 8.4，固液比为1:2.2，接种量7%，装液量7 L，发酵时间为50 h，发酵液最高芽孢数达到1.5×109 cfu/mL，最高蛋白质浓度0.69 mg/mL。 |
| 英文摘要 | Asparagus (Asparagus officinalis L.) is a nutritious and healthy vegetable cultivated as an important economic crop all over the temperate world. In some countries, it has been used as an anti-inflammatory, antifungal and anticancer herbal medicine for a long time. Asparagus spears are usually processed into canned products and vegetables in asparagus processing industry, while about 30～40% leftover bits and pieces of the raw materials will be discarded because of its more fibers. Tons of asparagus leftover bits and pieces cause environmental pollution and the useful resources waste. It has been determined that those wastes are rich in nutrients and active ingredients, the contents of some ingredients are even higher than those in vegetable asparagus. So, it is necessary to reuse the active substances from asparagus wastes through biorefinery method. In this study, asparagus chlorophyll was extracted and then sodium copper chlorophyll (SCC) was synthesized. Asparagus polysaccharide was isolated and purified to study its physicochemical property and biological activity. Finally, asparagus residue was used for the fermentation of Bacillus thuringiensis (Bt) biological pesticide. Therefore, the comprehensive utilization of asparagus waste resources was achieved. 1. The extraction process of chlorophyll and the preparation of sodium copper chlorophyllin from the leaves of asparagus were studied. The extraction conditions were as follows: the extractant was 80% acetone and 95% ethanol with the ratio of 1:4, the ratio of liquid to solid was 10:1, the extraction temperature was 70℃, the extraction time was 60 min and ultrasonic power was 160 W. The extraction rate of asparagus chlorophyll was 0.287%. Asparagus chlorophyll solution was unstable to light, temperature, acidic environment as well as oxidant, limiting its application, therefore appropriate chemical modification of chlorophyll was needed to prepare stable chlorophyll derivatives. The synthesis of sodium copper chlorophyllin was studied. The best saponification process parameters were pH 11, saponification time 60 min, saponification temperature 60 °C, the concentration multiple 5. The recovery of copper chlorophyllin acid should be treated by 100 W ultrasound for 30 seconds or 200 W ultrasound for 20 seconds. After the ultrasonic treatment, the product was recovered using filtration or centrifugation. Acidification and copper substitution reaction process was as follows: pH 3, time 2 h, temperature 70 °C, the actual amount of copper sulfate was 1.5 times of the theoretical amount, the product yield was 0.18% based on fresh leaves. The SCC product obtained was a dark green powder with metallic luster, slightly soluble in alcohol, chloroform and ether, soluble in water, easy to absorb moisture in the air. The sodium copper chlorophyllin's quality conformed to the national standard GB 3262-1982. Other byproducts were recovered. β-carotene was separated by Al2O3 column chromatography. β-carotene content in the fresh leaves was 0.0161%, and the recovery rate was 49.3%, purity was 90%. Asparagus polysaccharide was extracted from asparagus residue with the yield of 13.3%, and purity of 20.1%. Crude asparagus saponins were recovered from ethanol distillation with the yield of 12.3% and purity of 14.23 %. 2. Polysaccharides were extracted from Asparagus officinalis. A novel ultrasonic circulating extraction (UCE) technology was applied for the polysaccharide extraction. Three main factors (ultrasonic power, extracting time and liquid-solid ratio) were chosen for optimizing extraction parameters of Asparagus officinalis polysaccharides (AOP) by employing three-factors, three-levels Box-Behnken design (BBD). Five replicates at the centre of the design were used to estimate a pure error sum of squares. By employing multiple regression analysis based on the experimental data, the predicted response Y for the yield of polysaccharides can be obtained by the following second-order polynomial equation: Y=3.04 + 0.23X1 + 0.19X2 + 0.16X3 + 0.057X1X2 – 0.00425X1X3 – 0.06 X2X3 – 0.74 – 0.14 – 0.21 The optimal extraction conditions were as follows: ultrasonic power was 600 W, duty ratio 1.5:1, extraction time was 46 min, the liquid-solid ratio was 35 mL/g. Under these conditions, the experimental yield of polysaccharides was 3.134%, which was agreed closely to the predicted value. Compared with hot water extraction of AOP, ultrasonic circulating extraction (UCE) technique employed for the extraction of AOP in this study was time-saving, energy-saving and gave a higher yield. With hot water extraction method, the yield of AOP was just 2.253% at 60℃ for 120 min. On the other hand, lengthening extraction time excessively at high temperature (80℃) may induce the degradation of polysaccharides and therefore lead to the decline of AOP yield (2.151%). The crude polysaccharides were deproteinized and decolored by Sevage method and activated carbon. Three main polysaccharide fractions, AOP-4, AOP-6 and AOP-8 were obtained by fractional precipitation with gradient concentrations of ethanol (40%, 60% and 80%). The polysaccharides were composed of glucose, fucose, arabinose, galactose and rhamnose in a ratio of 2.18:1.86:1.50:0.98:1.53. Compared with hot water extraction (HWE), UCE showed time-saving, higher yield and no influence on the structure of asparagus polysaccharides. The results indicated that ultrasonic circulating extraction technology was an effective and advisable technique for the large scale production of plant polysaccharides. The in vitro antitumor and antioxidant activities of the polysaccharide fractions were evaluated by MTT assay and free radical-scavenging assay, respectively. Deproteinized AOPs showed higher antioxidant and antitumor activities than crude AOP. AOP-4 with molecular weight 5.75×104 Da showed significant function of scavenging hydroxyl radical. Three AOP fractions had significant antitumor activity against HeLa and BEL-7404 cells in a dose dependent manner. Furthermore, the inhibit activity of AOP-4 against HeLa cells was higher than those of other AOPs and the inhibition rate reached 83.96% at the concentration of 10 mg/mL. These results indicated that the AOP might be useful for developing natural safe antitumor drugs or health food. 3. Bt biological pesticide was produced by solid-state fermentation using asparagus extract residue. The optimal culture conditions were as follows: incubation temperature 30 °C, humidity 85%, initial pH 7.4, aeration rate ranged from 1 to 1.2 vvm. At this fermentation conditions, spore number was 1.55×1011 cfu/g of dry fermented material, the protein content was 8.83 mg/g of dry fermented material. The fermentation of Bt using asparagus waste in a 10 L mechanically stirring bioreactor was studied. The fermentation conditions were as follows: initial pH was 8.4, inoculation was 7%, the solid-liquid ratio was 1:2.2, culture time was 50 h. Under these conditions, the production of spores was 1.5×109 cfu/mL, the highest protein concentration was 0.69 mg/mL. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1867]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 赵庆生. 芦笋废弃资源的生物炼制[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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